POS0479 STAT3 DEGRADERS PROTECT FROM IMMUNOFIBROTIC CHANGES IN PRECLINICAL MODELS
Authors: P. Blyszczuk, G. Kania, E. Pachera, F. Rolski, A. Hukara, V. Tela, M. Mayo, V. Dixit, B. Yang, J. Gollob, N. Mainolfi, A. Slavin, C. Hubeau, O. Distler
Journal: Annals of the Rheumatic Diseases (2022)

Background The ubiquitin-proteasome system (UPS) is the endogenous intracellular mechanism for maintaining protein homeostasis through protein degradation and turnover. Heterobifunctional small molecules are a new class of compounds that form a ternary complex with an E3 ligase and protein of interest leading to ubiquitination and subsequent degradation of the protein of interest in a process known as Targeted Protein Degradation. This new therapeutic modality enables targeting of “undruggable” proteins such as STAT3, a transcription factor activated in immunofibrotic diseases. Objectives Kymera has developed heterobifunctional molecules that potently and selectively target STAT3 for degradation and elimination by the ubiquitin-proteasome pathway. The aim of these studies was to evaluate the therapeutic potential of pharmacologically removing STAT3 by targeted protein degradation in various human cell types in vitro, and to prevent the development of skin and lung fibrosis in vivo. Methods Dermal fibroblasts obtained from healthy and systemic sclerosis patients activated with TGF-β were analyzed for development of α-smooth muscle actin (α-SMA)-positive stress fibers and for contractility using collagen gel contraction assay. Contraction assay was also performed using human dermal smooth muscle cells. Human aortic endothelial cells (HAECs) were activated with LPS, and their adhesive properties were assessed in the microcapillary system by the ability to bind peripheral blood mononuclear cells (PBMCs) under shear stress. HAECs proliferation was induced with VEGF. THP-1 cells and CD14 + monocytes were activated with IL-6 or LPS, and secreted cytokines were assessed by CBA. PBMCs activated with LPS, IL-6, IL-21, or IL-23 alone (pSTAT3 induction), or with a combination of anti-CD3/CD38 beads and a pro-Th17 cocktail comprised of cytokines and antibodies to evaluate the development of a Th17 and Treg phenotype by flow cytometry. Cytokines were analyzed by ELISA. All cell types were pre-treated with STAT3 degraders 20h prior to experiment start. Intratracheal instillation of bleomycin was used to induce pulmonary fibrosis. Transgenic Tsk-1 mice were used as a model of spontaneous skin fibrosis. Results STAT3 degraders completely ablated STAT3 in all analyzed cell types with DC50 ranging from 0.25-0.8 nM. STAT3 degradation prevented TGF-β-induced formation of α-SMA-positive stress fibers in dermal fibroblasts (IC50 = 0.35nM) and 2 and 10nM degrader completely abrogated their contractility. Similarly, STAT3 degradation reduced the constitutive contractility of dermal smooth muscle cells of 13% (p&lt;0.05, n=6). Treatment of HAECs with STAT3 degraders resulted in anti-adhesive 178±21 for LPS and 93±12 for LPS +degrader, p&lt;0.05, n=6) and anti-proliferative 1.2±0.1 for VEGF and 0.95±0.1 for VEGF +degrader, p&lt;0.05, n=10-11) effects. In monocyte-focused assays (CD14+ monocytes and THP-1 cells), STAT3 degradation potently and dose-dependently inhibited IL-6 and LPS-induced pSTAT3 levels and the ensuing release of MCP-1/CCL2 (24.3±3.7 for LPS and 20.2±3.2 for LPS +degrader, p&lt;0.05, n=6). In CD4+ T lymphocytes, STAT3 degradation promoted a Treg phenotype and suppressed the development of Th17 cells. Systemic treatment in vivo showed that prophylactic STAT3 degradation (7 mg/kg twice a week, i.p.) reduced disease severity in the bleomycin-induced pulmonary fibrosis model (Ashcroft‘s score, 4.7±1.9 vs. 3.1±1.6, p&lt;0.05, n=11). In Tsk-1 mice that show co-occurrence of spontaneous skin thickening and robust STAT3 activation, STAT3 degrader treatment (2 or 7 mg/kg twice a week, i.p.) for 7 weeks significantly reduced thickness of the skin (701±238 vs. 480±205 vs. 365±107, p&lt;0.05, n=6-8). Conclusion STAT3 degraders that selectively and potently eliminate STAT3 show robust anti-inflammatory and anti-fibrotic potential in vitro and in vivo. Our results suggest that targeted protein degradation is a promising approach to modulate the STAT3 pathway, making it a novel therapeutic opportunity to treating multiple immunofibrotic diseases. Disclosure of Interests Przemyslaw Blyszczuk Grant/research support from: Kymera, Gabriela Kania: None declared, Elena Pachera: None declared, Filip Rolski: None declared, Amela Hukara: None declared, Vanessa Tela: None declared, Michele Mayo Employee of: Kymera, Vaishali Dixit Employee of: Kymera, Bin Yang Employee of: Kymera, Jared Gollob Employee of: Kymera, Nello Mainolfi Employee of: Kymera, Anthony Slavin Employee of: Kymera, Cedric Hubeau Employee of: Kymera, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim.